CEA及CK20的不同检测方法对结直肠癌诊断及预后评估的初步探讨
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摘要
背景
结直肠癌是最常见的消化道肿瘤之一,结直肠癌患者实行了根治性手术后,仍会有相当一部分的患者不可避免的出现复发,术后5年生存率一直徘徊在45%-55%之间。
细胞角蛋白20 (cytokertin20,CK20)是分布于外胚层起源细胞中的中间纤维丝,为细胞骨架的组成部分之一。CK20具有更为严格的上皮组织特异性。正常组织中,CK20见于肠粘膜细胞,胃粘膜及幽门腺体细胞,十二指肠粘膜细胞,泌尿系伞状细胞,表皮M erkel细胞,而其它组织均为阴性。CK20在细胞发生化生、恶变、肿瘤转移、体外培养等异常时,这种表达持续存在。癌胚抗原(carcinoembryonic antigen, CEA)是一种重要的肿瘤相关抗原和国际公认的肿瘤标记物,相对分子质量为180,000的胚胎性癌蛋自,广泛地存在于上皮源性恶性肿瘤组织中,在90%的胃肠道癌,60%的胰腺癌、50%的乳腺癌,70%的非小细胞肺癌中有较高的表达。
目的
对CEA和CK20在结直肠癌不同组织的表达及它们之间的关系做出初步研究,探讨两者与结直肠癌临床和病理之间的关系。
方法
采用免疫组织化学法分别检测CEA和CK20蛋白在结直肠癌患者的癌组织、癌旁组织、肠系膜淋巴结组织及正常肠粘膜组织中的表达。
结果
CEA和CK20蛋白在组织中的表达定位于细胞浆和细胞膜。除正常对照组为阴性外其余各组CEA蛋白均呈不同程度的表达。CEA在癌组织中的总阳性表达率为92.86%；癌旁组织组CEA的表达高于肠系膜淋巴结组。包括正常对照组在内各组CK20蛋白均有不同程度的表达,癌组织组CK20的表达高于癌旁组织组,癌旁组织组的表达高于正常对照组的表达,正常对照组的表达高于肠系膜淋巴结组。
结论
1、CEA在各种不同结直肠癌组织中均有不同程度的表达,正常结肠粘膜无表达。其在癌组织中的表达与肿瘤的Dukes分期有关。
2、CK20在正常结肠粘膜及结直肠癌组织中均有不同程度的表达,其在癌组织中的表达与有无淋巴结转移有关。
背景
结直肠癌出现转移和复发与肿瘤的隐性微转移(occult micrometastases)及循环肿瘤细胞(circulating tumor cells, CTCS)密切相关,目前常用检测CTCS的技术有免疫细胞化学法、逆转录聚合酶链反应、流式细胞术等方法。由于这些方法或较费时、或程序复杂、或检查结果假阳性率高,难以推广应用于临床。已经出现一些新的方法,如实时荧光定量PCR、蛋白指纹图谱技术等。但以上的方法实验成本较高,目前还难以适应临床的实际需要,近年来,免疫传感器分析法由于其灵敏度高、选择性好和结构简单等特点而得到了越来越广泛的重视。免疫传感器是基于检测抗原-抗体结合的一种高选择性生物传感器。本实验首次采用免疫传感器分析法检测结直肠癌患者血清中CEA、CK20的表达。
目的
初步探讨准确性、特异性高且简便实用的CEA、CK20检测方法以期改善结直肠癌诊断及预后评估。
方法
分别采用酶联免疫法、实时荧光定量PCR及免疫传感器分析法序列性联合检测结直肠癌患者不同组织及外周血中CEA及CK20的表达,以探讨其在结直肠癌的诊断及预后评估中的价值。
结果
实时荧光定量PCR技术结合免疫传感器及ELISA等方法对结直肠癌患者外周血CEA、CK20及不同组织CEAmRNA、CK20mRNA进行检测发现：采用灵敏度较高的realtime-PCR有利于提高基因检测阳性率,尤其是外周血CEAmRNA的阳性率从57.14%提高到89.28%；Dukes A, B期的15例患者中,通过CK20的免疫组化染色发现有3例患者(74个淋巴结)有微转移,进一步检测CK20mRNA的拷贝表达发现有9例患者(155个淋巴结)有微转移；CEA和CK20免疫传感器应用于临床标本的检测结果与ELISA法比较无显著性差异,尽管灵敏度和准确性不及realtime-PCR,但特异性要强于realtime-PCR,且其阳性率还高于ELISA法,CEA和CK20免疫传感器能用于血清CEA的定量检测。
结论
一、改进检测方法能提高结直肠癌患者外周血CEA、CK20的阳性率,能为诊断及预后评估提供帮助。
二、realtime-PCR检测肠系膜淋巴结CK20mRNA的表达对确定结直肠癌患者的分期有重要的价值,对预后的评估也有一定意义。
三、首次成功建立CEA、CK20的多巴胺化学氧化原位包埋法免疫传感器,并应用于检测结直肠癌患者外周血CEA、CK20。
背景
决定结直肠癌患者预后的最重要指标是肿瘤的浸润深度与淋巴结转移的程度。但是,在部分实施了根治性手术的结直肠癌患者中,经病理学常规检查未发现淋巴结转移者仍会发生转移和复发,有研究认为该现象与结直肠癌淋巴结微转移有关。
1992年UICC将隐匿性转移(occult metastasis)或微转移(micrometastasis, MM)限定为单个转移肿瘤细胞或转移肿瘤细胞团直径<2 mm。多数文献报告将常规组织学检查未发现转移灶而经免疫组化或分子生物学技术检测发现的淋巴结转移灶定义为淋巴结微转移。因此,淋巴结微转移目前尚无统一的标准。
本研究的前面两部分实验已经证明采用免疫组化、ELISA、免疫传感器、realtime-PCR等方法检测结直肠癌患者外周血及肠系膜淋巴结中CEA、CK20及CEAmRNA、CK20mRNA,结果发现循环血液微转移灶及淋巴结微转移灶。
目的
探讨CEA、CK20以及CEAmRNA和CK20mRNA与微转移的关系,了解其能否成为结直肠癌分期及预后评估的有效方法。
方法
通过临床随访及临床应用统计学方法对两组测定值进行比较分析。
结果
影响预后的指标有：外周血CK20阳性率、淋巴结CK20mRNA阳性率、淋巴结CEAmRNA阳性率、术前合并梗阻或穿孔、糖尿病、肿瘤浸润深度、下切缘与肿瘤距离、送检淋巴结数目、有无淋巴结转移、术后有无化疗(p<0.05)。其中淋巴结CK20mRNA阳性率、淋巴结CEAmRNA阳性率、术前合并梗阻或穿孔、肿瘤浸润深度、下切缘与肿瘤距离是影响结直肠癌预后的独立因素,而外周血CK20阳性率、术后有无化疗、糖尿病、送检淋巴结数目则未能进入Cox多因素模型。
结论采用realtime-PCR的方法检测肠系膜淋巴结的CK20mRNA表达在结直肠癌诊断及预后评估中有一定的意义和价值。
Background
Colorectal cancer is one of the most common carcinomas in the digestive tract. There will be a considerable part of patients who underwent a curative resection are going to encounter the situation of tumor recurrence. The 5-year survival rates ranged between 45%-55%.
Cytokertin 20(CK20) is a kind of intermediate filament that distributes original cells of the ectoderm, it participates making up the cytoskeleton. CK20 presents a more restrict specificity of epithelial cells. Cytokertin20 is found in nowhere but cells in intestinal mucosa、duodenum mucosa、gastric mucosa, in gland cells in pylorus, in umbrella cells of the urinary system, in merkel cells of epidermis, et al. Cytokertin20 also continually expresses when metaplasia, malignant, metastasis happens or external cultivation. Carcinoembryonic antigen (CEA) is a kind of significant tumor-associated antigen and tumor marker, that is internationally recognized. It is a embryos of oncoprotein weights 180,000 D. CEA expresses widely in epithelial carcinoma, and expresses highly in 90%gastrointesinal cancer,50%breast cancer and 70%non-small cell lung cancer.
Objective
To investigate the expression and relationships of CEA and CK20 in different tissues of colorectal carcinoma preliminarily, and to investigate the relationship between CEA、CK20 expression and clinical characteristics of patients of colorectal cancer.
Methods
Detect the expression of CEA and CK20 respectively using ways of immunohistochemistry in cancer tissue, adjacent normal mucosa, normal mucosa and mesenteric lymph node tissue.
Results
The expression of CEA and CK20 locates in cytoplasm and membrane of epithelial cells. There is a varying degrees expression of CEA in all the cancer tissue, adjacent normal mucosa, and mesenteric lymph node tissue, however, in normal mucosa, as control group, CEA expresses negatively. The total positive rate of CEA expression in cancer tissue is 92.86%; CEA expresses higher in adjacent normal mucosa than mesenteric lymph node tissue. CK20 expresses verily in every group including in normal mucosa, CK20 expresses higher in cancer tissue than in adjacent normal mucosa, expresses higher in adjacent normal mucosa than in normal mucosa, the mesenteric lymph node tissue has a lowest CK20 expression.
Conclusion
1、CEA expresses in varying degrees in different colorectal cancer tissue, but not in normal mucosa. There is a certain relationship between Dukes stage and CEA expression in cancer tissue.
2、CK20 expresses in varying degrees in all groups including normal mucosa and different colorectal cancer tissue, and the degree of CK20 expression concerns lymph node metastasis status.
Background
The metastasis and recurrence of colorectal cancer is closely related to occult micro metastases and circulating tumor cells (CTCS). Such as immunocytochemistry、reverse transcription polymerase chain reaction, flow cytometry, et al. which are usually used for detecting CTCS. However, those methods are either time-consuming or complicated or even result in a false positive outcome, so they are hard to be used in clinical work extensively. There are some new methods have emerged already, for example:real-time fluorescence quantitative PCR, protein fingerprinting technology, nevertheless, these technologies still can't meet the request in daily clinical work because of they are too costly. Immunosensor analysis is applied more and more extensively in recent years, because it's sensitivity, selectivity and simple. Immunosensor is a kind of highly selective biosensor that based on the detection of antigen-antibody binding. Our experiment applied Immunosensor analysis to detect expression of CEA and CK20 in serum of patients of colorectal cancer for the first time.
Objective
To investigate a determine system for judging the status and predicting the prognosis of colorectal cancer.
Methods
Use ICC、ELISA、real-time fluorescence quantitative PCR and immunosensor analysis to detect the expression of CEA and CK20 in colorectal cancer, respectively, to determine the value of immunosensor analysis in judging the status and predicting the prognosis of colorectal cancer.
Results
Use real-time fluorescence quantitative PCR、immunosensor analysis and ELISA to detect CEA、CK20 in patients'peripheral blood, and to detect CEAmRNA、CK20mRNA in different tissue of colorectal cancer shows that real-time fluorescence quantitative PCR is more sensitive and helpful in improving the positive rate of genetic testing:the positive rate of CEAmRNA in peripheral blood is raised from 57.14% to 89.28%. In 15 patients who are in Dukes A、B,3 patients(741ymph notes) were found to have micro-metastasis through testing CK20 using immunohistochemistry way, testing CK20mRNA copies found 9 more patients(1551ymph notes) to have micro-metastasis. Although the sensitivity and accuracy is not as good as realtime-PCR, the using of immunosensor of CEA and CK20 in detecting clinical specimens results in an outcome that has no significant differences compared with ELISA way, but immunohistochemistry is more specific than realtime-PCR, and results in a higher positive rate than ELISA. Immunosensor of CEA and CK20 can be used to detect CEA quantitatively in serum.
Conclusion
1、Improving way of detecting CEA、CK20 in patients'peripheral blood of colorectal cancer can achieve a higher positive rate, and going to help effectively diagnosing colorectal cancer and predicting it's prognosis.
2、There is great value for realtime-PCR in testing the expression of CK20mRNA of mesenteric lymph nodes in judging the exact status and in predicting the prognosis of colorectal cancer.
3、The Embedding method in situ chemical oxidation of dopamine method is established successfully for CEA and CK20 immunosensor, and for testing CEA、CK20 in patients'peripheral blood of colorectal cancer.
Background
The most important indexes which conclude the prognosis of colorectal cancer are the infiltrating depth of the tumor and the lymph notes metastasis degree. However, in some colorectal cancer patients who are treated radical surgery with no lymph notes metastasis by pathology routine examination, metastasis and recurrence happen. It is studied that it is related to lymph nodes micro-metastasis of colorectal cancer.
In 1992, UICC define occult metastasis and micro-metastasis (MM) as the diameter of single metastatic tumor cell or metastatic tumor cell clump less than 2mm. Major literatures and reports define lymph nodes metastasis focus which are discovered by immune histochemistry instead of routine histological examination. Thus, there is no unified standard in defining lymph nodes micro-metastasis.
The former two parts of experiment in this research have proved we can find out occult metastasis and micro-metastasis by detecting CEA、lymph notes of colorectal cancer patients.
Objective
To investigate what is the relationship between CEA、CK20 and CEAmRNA、CK20mRNA, and if they can be used as independent factors in deciding status and prognosis of colorectal cancer of assessment system.
Methods
By clinical follow-up and clinical application of statistical methods to compare the two groups of measured values.
Results
Prognostic indicators include:The positive rate of CK20 in peripheral blood, lymph node positive rate of CK20mRNA, lymph node positive rate of CEAmRNA, Preoperative combined obstruction or perforation, diabetes, tumor invasion depth, distance from the tumor lower margin,the number of lymph nodes under examination, lymph node metastasis after operation with or without chemotherapy (p<0.05). Lymph node positive rate of CK20mRNA, lymph node positive rate of CEAmRNA, preoperative combined obstruction or perforation, tumor invasion depth, distance from the tumor lower margin were independent prognostic factors in colorectal cancer. The positive rate of CK20 in peripheral blood, diabetes, with or without chemotherapy after surgery and the number of lymph nodes under examination were unable to enter the Cox multivariate model.
Conclusion
The application of realtime-PCR to detect the expression of CK20mRNA in mesenteric lymph nodes in diagnosis and prognosis system of colorectal cancer is of great significance and value.
结直肠癌是最常见的消化道肿瘤之一,结直肠癌患者实行了根治性手术后,仍会有相当一部分的患者不可避免的出现复发,术后5年生存率一直徘徊在45%-55%之间。
细胞角蛋白20 (cytokertin20,CK20)是分布于外胚层起源细胞中的中间纤维丝,为细胞骨架的组成部分之一。CK20具有更为严格的上皮组织特异性。正常组织中,CK20见于肠粘膜细胞,胃粘膜及幽门腺体细胞,十二指肠粘膜细胞,泌尿系伞状细胞,表皮M erkel细胞,而其它组织均为阴性。CK20在细胞发生化生、恶变、肿瘤转移、体外培养等异常时,这种表达持续存在。癌胚抗原(carcinoembryonic antigen, CEA)是一种重要的肿瘤相关抗原和国际公认的肿瘤标记物,相对分子质量为180,000的胚胎性癌蛋自,广泛地存在于上皮源性恶性肿瘤组织中,在90%的胃肠道癌,60%的胰腺癌、50%的乳腺癌,70%的非小细胞肺癌中有较高的表达。
目的
对CEA和CK20在结直肠癌不同组织的表达及它们之间的关系做出初步研究,探讨两者与结直肠癌临床和病理之间的关系。
方法
采用免疫组织化学法分别检测CEA和CK20蛋白在结直肠癌患者的癌组织、癌旁组织、肠系膜淋巴结组织及正常肠粘膜组织中的表达。
结果
CEA和CK20蛋白在组织中的表达定位于细胞浆和细胞膜。除正常对照组为阴性外其余各组CEA蛋白均呈不同程度的表达。CEA在癌组织中的总阳性表达率为92.86%；癌旁组织组CEA的表达高于肠系膜淋巴结组。包括正常对照组在内各组CK20蛋白均有不同程度的表达,癌组织组CK20的表达高于癌旁组织组,癌旁组织组的表达高于正常对照组的表达,正常对照组的表达高于肠系膜淋巴结组。
结论
1、CEA在各种不同结直肠癌组织中均有不同程度的表达,正常结肠粘膜无表达。其在癌组织中的表达与肿瘤的Dukes分期有关。
2、CK20在正常结肠粘膜及结直肠癌组织中均有不同程度的表达,其在癌组织中的表达与有无淋巴结转移有关。
背景
结直肠癌出现转移和复发与肿瘤的隐性微转移(occult micrometastases)及循环肿瘤细胞(circulating tumor cells, CTCS)密切相关,目前常用检测CTCS的技术有免疫细胞化学法、逆转录聚合酶链反应、流式细胞术等方法。由于这些方法或较费时、或程序复杂、或检查结果假阳性率高,难以推广应用于临床。已经出现一些新的方法,如实时荧光定量PCR、蛋白指纹图谱技术等。但以上的方法实验成本较高,目前还难以适应临床的实际需要,近年来,免疫传感器分析法由于其灵敏度高、选择性好和结构简单等特点而得到了越来越广泛的重视。免疫传感器是基于检测抗原-抗体结合的一种高选择性生物传感器。本实验首次采用免疫传感器分析法检测结直肠癌患者血清中CEA、CK20的表达。
目的
初步探讨准确性、特异性高且简便实用的CEA、CK20检测方法以期改善结直肠癌诊断及预后评估。
方法
分别采用酶联免疫法、实时荧光定量PCR及免疫传感器分析法序列性联合检测结直肠癌患者不同组织及外周血中CEA及CK20的表达,以探讨其在结直肠癌的诊断及预后评估中的价值。
结果
实时荧光定量PCR技术结合免疫传感器及ELISA等方法对结直肠癌患者外周血CEA、CK20及不同组织CEAmRNA、CK20mRNA进行检测发现：采用灵敏度较高的realtime-PCR有利于提高基因检测阳性率,尤其是外周血CEAmRNA的阳性率从57.14%提高到89.28%；Dukes A, B期的15例患者中,通过CK20的免疫组化染色发现有3例患者(74个淋巴结)有微转移,进一步检测CK20mRNA的拷贝表达发现有9例患者(155个淋巴结)有微转移；CEA和CK20免疫传感器应用于临床标本的检测结果与ELISA法比较无显著性差异,尽管灵敏度和准确性不及realtime-PCR,但特异性要强于realtime-PCR,且其阳性率还高于ELISA法,CEA和CK20免疫传感器能用于血清CEA的定量检测。
结论
一、改进检测方法能提高结直肠癌患者外周血CEA、CK20的阳性率,能为诊断及预后评估提供帮助。
二、realtime-PCR检测肠系膜淋巴结CK20mRNA的表达对确定结直肠癌患者的分期有重要的价值,对预后的评估也有一定意义。
三、首次成功建立CEA、CK20的多巴胺化学氧化原位包埋法免疫传感器,并应用于检测结直肠癌患者外周血CEA、CK20。
背景
决定结直肠癌患者预后的最重要指标是肿瘤的浸润深度与淋巴结转移的程度。但是,在部分实施了根治性手术的结直肠癌患者中,经病理学常规检查未发现淋巴结转移者仍会发生转移和复发,有研究认为该现象与结直肠癌淋巴结微转移有关。
1992年UICC将隐匿性转移(occult metastasis)或微转移(micrometastasis, MM)限定为单个转移肿瘤细胞或转移肿瘤细胞团直径<2 mm。多数文献报告将常规组织学检查未发现转移灶而经免疫组化或分子生物学技术检测发现的淋巴结转移灶定义为淋巴结微转移。因此,淋巴结微转移目前尚无统一的标准。
本研究的前面两部分实验已经证明采用免疫组化、ELISA、免疫传感器、realtime-PCR等方法检测结直肠癌患者外周血及肠系膜淋巴结中CEA、CK20及CEAmRNA、CK20mRNA,结果发现循环血液微转移灶及淋巴结微转移灶。
目的
探讨CEA、CK20以及CEAmRNA和CK20mRNA与微转移的关系,了解其能否成为结直肠癌分期及预后评估的有效方法。
方法
通过临床随访及临床应用统计学方法对两组测定值进行比较分析。
结果
影响预后的指标有：外周血CK20阳性率、淋巴结CK20mRNA阳性率、淋巴结CEAmRNA阳性率、术前合并梗阻或穿孔、糖尿病、肿瘤浸润深度、下切缘与肿瘤距离、送检淋巴结数目、有无淋巴结转移、术后有无化疗(p<0.05)。其中淋巴结CK20mRNA阳性率、淋巴结CEAmRNA阳性率、术前合并梗阻或穿孔、肿瘤浸润深度、下切缘与肿瘤距离是影响结直肠癌预后的独立因素,而外周血CK20阳性率、术后有无化疗、糖尿病、送检淋巴结数目则未能进入Cox多因素模型。
结论采用realtime-PCR的方法检测肠系膜淋巴结的CK20mRNA表达在结直肠癌诊断及预后评估中有一定的意义和价值。
Background
Colorectal cancer is one of the most common carcinomas in the digestive tract. There will be a considerable part of patients who underwent a curative resection are going to encounter the situation of tumor recurrence. The 5-year survival rates ranged between 45%-55%.
Cytokertin 20(CK20) is a kind of intermediate filament that distributes original cells of the ectoderm, it participates making up the cytoskeleton. CK20 presents a more restrict specificity of epithelial cells. Cytokertin20 is found in nowhere but cells in intestinal mucosa、duodenum mucosa、gastric mucosa, in gland cells in pylorus, in umbrella cells of the urinary system, in merkel cells of epidermis, et al. Cytokertin20 also continually expresses when metaplasia, malignant, metastasis happens or external cultivation. Carcinoembryonic antigen (CEA) is a kind of significant tumor-associated antigen and tumor marker, that is internationally recognized. It is a embryos of oncoprotein weights 180,000 D. CEA expresses widely in epithelial carcinoma, and expresses highly in 90%gastrointesinal cancer,50%breast cancer and 70%non-small cell lung cancer.
Objective
To investigate the expression and relationships of CEA and CK20 in different tissues of colorectal carcinoma preliminarily, and to investigate the relationship between CEA、CK20 expression and clinical characteristics of patients of colorectal cancer.
Methods
Detect the expression of CEA and CK20 respectively using ways of immunohistochemistry in cancer tissue, adjacent normal mucosa, normal mucosa and mesenteric lymph node tissue.
Results
The expression of CEA and CK20 locates in cytoplasm and membrane of epithelial cells. There is a varying degrees expression of CEA in all the cancer tissue, adjacent normal mucosa, and mesenteric lymph node tissue, however, in normal mucosa, as control group, CEA expresses negatively. The total positive rate of CEA expression in cancer tissue is 92.86%; CEA expresses higher in adjacent normal mucosa than mesenteric lymph node tissue. CK20 expresses verily in every group including in normal mucosa, CK20 expresses higher in cancer tissue than in adjacent normal mucosa, expresses higher in adjacent normal mucosa than in normal mucosa, the mesenteric lymph node tissue has a lowest CK20 expression.
Conclusion
1、CEA expresses in varying degrees in different colorectal cancer tissue, but not in normal mucosa. There is a certain relationship between Dukes stage and CEA expression in cancer tissue.
2、CK20 expresses in varying degrees in all groups including normal mucosa and different colorectal cancer tissue, and the degree of CK20 expression concerns lymph node metastasis status.
Background
The metastasis and recurrence of colorectal cancer is closely related to occult micro metastases and circulating tumor cells (CTCS). Such as immunocytochemistry、reverse transcription polymerase chain reaction, flow cytometry, et al. which are usually used for detecting CTCS. However, those methods are either time-consuming or complicated or even result in a false positive outcome, so they are hard to be used in clinical work extensively. There are some new methods have emerged already, for example:real-time fluorescence quantitative PCR, protein fingerprinting technology, nevertheless, these technologies still can't meet the request in daily clinical work because of they are too costly. Immunosensor analysis is applied more and more extensively in recent years, because it's sensitivity, selectivity and simple. Immunosensor is a kind of highly selective biosensor that based on the detection of antigen-antibody binding. Our experiment applied Immunosensor analysis to detect expression of CEA and CK20 in serum of patients of colorectal cancer for the first time.
Objective
To investigate a determine system for judging the status and predicting the prognosis of colorectal cancer.
Methods
Use ICC、ELISA、real-time fluorescence quantitative PCR and immunosensor analysis to detect the expression of CEA and CK20 in colorectal cancer, respectively, to determine the value of immunosensor analysis in judging the status and predicting the prognosis of colorectal cancer.
Results
Use real-time fluorescence quantitative PCR、immunosensor analysis and ELISA to detect CEA、CK20 in patients'peripheral blood, and to detect CEAmRNA、CK20mRNA in different tissue of colorectal cancer shows that real-time fluorescence quantitative PCR is more sensitive and helpful in improving the positive rate of genetic testing:the positive rate of CEAmRNA in peripheral blood is raised from 57.14% to 89.28%. In 15 patients who are in Dukes A、B,3 patients(741ymph notes) were found to have micro-metastasis through testing CK20 using immunohistochemistry way, testing CK20mRNA copies found 9 more patients(1551ymph notes) to have micro-metastasis. Although the sensitivity and accuracy is not as good as realtime-PCR, the using of immunosensor of CEA and CK20 in detecting clinical specimens results in an outcome that has no significant differences compared with ELISA way, but immunohistochemistry is more specific than realtime-PCR, and results in a higher positive rate than ELISA. Immunosensor of CEA and CK20 can be used to detect CEA quantitatively in serum.
Conclusion
1、Improving way of detecting CEA、CK20 in patients'peripheral blood of colorectal cancer can achieve a higher positive rate, and going to help effectively diagnosing colorectal cancer and predicting it's prognosis.
2、There is great value for realtime-PCR in testing the expression of CK20mRNA of mesenteric lymph nodes in judging the exact status and in predicting the prognosis of colorectal cancer.
3、The Embedding method in situ chemical oxidation of dopamine method is established successfully for CEA and CK20 immunosensor, and for testing CEA、CK20 in patients'peripheral blood of colorectal cancer.
Background
The most important indexes which conclude the prognosis of colorectal cancer are the infiltrating depth of the tumor and the lymph notes metastasis degree. However, in some colorectal cancer patients who are treated radical surgery with no lymph notes metastasis by pathology routine examination, metastasis and recurrence happen. It is studied that it is related to lymph nodes micro-metastasis of colorectal cancer.
In 1992, UICC define occult metastasis and micro-metastasis (MM) as the diameter of single metastatic tumor cell or metastatic tumor cell clump less than 2mm. Major literatures and reports define lymph nodes metastasis focus which are discovered by immune histochemistry instead of routine histological examination. Thus, there is no unified standard in defining lymph nodes micro-metastasis.
The former two parts of experiment in this research have proved we can find out occult metastasis and micro-metastasis by detecting CEA、lymph notes of colorectal cancer patients.
Objective
To investigate what is the relationship between CEA、CK20 and CEAmRNA、CK20mRNA, and if they can be used as independent factors in deciding status and prognosis of colorectal cancer of assessment system.
Methods
By clinical follow-up and clinical application of statistical methods to compare the two groups of measured values.
Results
Prognostic indicators include:The positive rate of CK20 in peripheral blood, lymph node positive rate of CK20mRNA, lymph node positive rate of CEAmRNA, Preoperative combined obstruction or perforation, diabetes, tumor invasion depth, distance from the tumor lower margin,the number of lymph nodes under examination, lymph node metastasis after operation with or without chemotherapy (p<0.05). Lymph node positive rate of CK20mRNA, lymph node positive rate of CEAmRNA, preoperative combined obstruction or perforation, tumor invasion depth, distance from the tumor lower margin were independent prognostic factors in colorectal cancer. The positive rate of CK20 in peripheral blood, diabetes, with or without chemotherapy after surgery and the number of lymph nodes under examination were unable to enter the Cox multivariate model.
Conclusion
The application of realtime-PCR to detect the expression of CK20mRNA in mesenteric lymph nodes in diagnosis and prognosis system of colorectal cancer is of great significance and value.
引文
1. Ghafoor A, Jemal A, Ward E, et al. Trends in breast cancer by race and ethnicity. CA Cancer J Clin 2003;53:342-355.
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4. Ward E, Jemal A, Cokkinides V, et al. Cancer disparities by race/ethnicity and socioeconomic status. CA Cancer J Clin 2004;54:78-93.
5. Ghafoor A, Jemal A, Cokkinides V, et al. Cancer statistics for African Americans. CA Cancer J Clin 2002;52:326-341.
6. Park JG. Cancer Incidence in Korea 1999-2001. Goyang:National Cancer Center; 2005.
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9. Kronberg U, Lopez-Kostner F, Soto G, Zuniga A, Wistuba I, Miranda V, et al. Detection of lymphatic micrometastases in patients with stages I and II colorectal cancer:impact on five-year survival. Dis Colon Rectum.2004;47:1151-7.
10. Rosenberg R, Friederichs J, Gertler R, Hoos A, Mueller J, Nahrig J, et al. Prognostic evaluation and review of immunohistochemically detected disseminated tumor cells in peritumoral lymph nodes of patients with pN0 colorectal cancer. Int J Colorectal Dis.2004; 19:430-7.
11. Tschmelitsch J, Klimstra DS, Cohen AM. Lymph node micrometastases do not predict relapse in stage II colon cancer. Ann Surg Oncol.2000;7:601-8.
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13. Noura S, Yamamoto H, Ohnishi T, Masuda N, Matsumoto T, Takayama O, et al. Comparative detection of lymph node micrometastases of stage II colorectal cancer by reverse transcriptase polymerase chain reaction and immunohistochemistry. J Clin Oncol.2002;20:4232-41.
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19. Wong JH, Johnson DS, Hemmings D, et al. Assessing the quality of colorectal cancer staging:documenting the process in improving the staging of node-negative colorectal cancer. Arch Surg.2005;140:881-887.
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1. Ghafoor A, Jemal A, Ward E, et al. Trends in breast cancer by race and ethnicity. CA Cancer J Clin 2003;53:342-355.
2. National Cancer Institute. Overview of the SEER Program. Available on the World Wide Web at http://seer.cancer.gov/about/. Accessed March 29,2006.
3. Nelson H, Petrelli N, Carlin A, et al. Guidelines 2000 for colon and rectal cancer surgery. J Natl Cancer Inst.2001;93:583-596.
4. Ward E, Jemal A, Cokkinides V, et al. Cancer disparities by race/ethnicity and socioeconomic status. CA Cancer J Clin 2004;54:78-93.
5. Ghafoor A, Jemal A, Cokkinides V, et al. Cancer statistics for African Americans. CA Cancer J Clin 2002;52:326-341.
6. Park JG. Cancer Incidence in Korea 1999-2001. Goyang:National Cancer Center; 2005.
7. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics:2007. CA Cancer J Clin.2007; 57:43-66.
8. Chen SL, Bilchik AJ. More extensive nod al dissection improves survival for stages I to III of colon cancer:a population-based study. Ann Surg.2006;244: 602-10.
9. Kronberg U, Lopez-Kostner F, Soto G, Zuniga A, Wistuba I, Miranda V, et al. Detection of lymphatic micrometastases in patients with stages I and II colorectal cancer:impact on five-year survival. Dis Colon Rectum.2004;47:1151-7.
10. Rosenberg R, Friederichs J, Gertler R, Hoos A, Mueller J, Nahrig J, et al. Prognostic evaluation and review of immunohistochemically detected disseminated tumor cells in peritumoral lymph nodes of patients with pN0 colorectal cancer. Int J Colorectal Dis.2004;19:430-7.
11. Tschmelitsch J, Klimstra DS, Cohen AM. Lymph node micrometastases do not predict relapse in stage II colon cancer. Ann Surg Oncol.2000;7:601-8.
12. Greene FL, Page DL, Fleming ID, Fritz A, Balch CM, Haller DG, et al. AJCC Cancer Staging Manual,6th ed. New York:Springer-Verlag; 2002. p.226-8.
13. Noura S, Yamamoto H, Ohnishi T, Masuda N, Matsumoto T, Takayama O, et al. Comparative detection of lymph node micrometastases of stage II colorectal cancer by reverse transcriptase polymerase chain reaction and
immunohistochemistry. J Clin Oncol.2002;20:4232-41.
14. Bilchik AJ, Hoon DS, Saha S, Turner RR, Wiese D, DiNome M, et al. Prognostic impact of micrometastases in colon cancer:interim results of a prospective multicenter trial. Ann Surg.2007;246:568-75.
15. Benson AB 3rd, Schrag D, Somerfield MR, Cohen AM, Figueredo AT, Flynn PJ, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol.2004;22:3408-19.
16. Sun Jin Park, M.D.1, Kil Yeon Lee, M.D.1, PhD. and Si Young Kim, M.D., Ph.D.2 Clinical Significance of Lymph Node Micrometastasis in Stage I and II Colon Cancer, Cancer Res Treat.2008; 40(2):75-80.
17. Andre'T, Boni C, Mounedji-Boudiaf L, et al. Oxaliplatin fluorouracil and leucovorin as adjuvant treatment for colon cancer. N Engl J Med.2004; 350:2343-2351.
18. Baxter NN, Virnig DJ, Rothenberger DA, et al. Lymph node evaluation in colorectal cancer patients:a population-based Study. J Natl Cancer Inst.2005; 95:219-225.
19. Wong JH, Johnson DS, Hemmings D, et al. Assessing the quality of colorectal cancer staging:documenting the process in improving the staging of node-negative colorectal cancer. Arch Surg.2005; 140:881-887.
20. Truong PT, Berthelet E, Lee J, et al. The prognostic significance of the percentage of positive/dissected axillary lymph nodes in breast cancer recurrence and survival in patients with one to three positive axillary nodes. Cancer. 2005;103:2006-2014.
21. Kim J, Cheong JH, Hyung WJ, et al. Predictors of long-term survival in pN3 gastric cancer patients. J Surg Oncol.2004;88:9-13.
22. Rodriguez Santiago JM, Munoz E, Marti M, et al. Metastatic lymph node ratio as a prognostic factor in gastric cancer. Eur J Surg Oncol.2005;31:59-66.
23. Berger AC, Sigurdson ER, LeVoyer T, et al. Colon cancer survival is associated with decreasing ratio of metastatic to examined lymph nodes. J Clin Oncol. 2005;23:8706-8712.
24. Jessup JM, Stewart A, Greene FL, et al. Adjuvant chemotherapy for stage III colon cancer:implications of race/ethnicity, age, and differentiation. JAMA.2005; 294:2703-2711.
25. Jemal A, Siegel R, Ward E, et al. Cancer statistics 2006. CA CancerJ Clin. 2006;56:106-130.
26. Swanson RS, Compton CC, Stewart AK, et al. The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined. Ann Surg Oncol. 2003; 10:65-71.
27. Joseph NE, Sigurdson ER, Hanlon AL, et al. Accuracy of determining nodal negativity in colorectal cancer on the basis of the number of nodes retrieved on resection. Ann Surg Oncol.2003; 10:213-218.
28. Tiwari RC, Ghosh K, Jemal A, et al. A new method of predicting US and state-level cancer mortality counts for the current calendar year. CA Cancer J Clin 2004;54:30-40.
29. Oyama K, Terashima M, Takagane A, Maesawa C. Prognostic significance of peritoneal minimal residual disease in gastric cancer detected by reverse transcription-polymerase chain reaction. Br J Surg 2004;91:435-43..
30. Ozbas S, Dafydd H, Purushotham AD. Bone marrow micrometastasis in breast cancer. Br J Surg 2003;90:290-301.
31. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 2005;353:793-802.
32. Macadam R, Sarela A, Wilson J, MacLennan K, Guillou P. Bone marrow micrometastases predict early post-operative recurrence following surgical resection of oesophageal and gastric carcinoma. Eur J Surg Oncol 2003;29:450-4.
33. Seeliger H, Spatz H, Jauch KW. Minimal residual disease in gastric cancer. Recent Results Cancer Res 2003; 162:79-87.
34. Barak V, Goitre H, Panaretakis KW, et al. Clinical utility of cytokeratins as tumor markers[J]Clin Biochem.2004.37(7):529-540
35. Iwaya K.Mukai K. Accumulation of ubiquitin-conjugated cytokeratin fragments in tumor cells[J]Semin Cancer Biol.2005,15(4):309-318.
36. Alessandro R.Belluco C, Kohn E C. Proteomic approaches in colon cancer: promising tools for new cancermarkers and drugtarget discovery [J].Clin Colorectal Cancer.2005-4(6)396-402.
37. Chen Y D, Zheng S, Yu J K, et al. Artificial neural networks analysis of surface-enhanced laser desorption/ionisation mass spectra of serum protein
pattern distinguishes colorectal cancer from healthy population [J].Clin Cancer Res,2004,10(24):8380-8385.
38. Ward D G, Suggett N, Cheng Y, et al. Identification of serum biomarkers for colon cancer by proteomic analysis [J]Br J Cancer.2006.94(12):1898-1905.
39. Albrethsen J, Bogebo R, Cammeltofr S, et al. Upregulated expression of human neutrophil peptides 1.2 and 3 (HNP 1-3) in colon cancer serum and tumours:a biomarker study [J]. BMC Cancer.2005.5:8.
40. Turck N, Richest S, Cendry P, et al. Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells [J].Proteomics,2004,4(1):93-105.
41. Carpelan-Holmstrom M, Louhimo J, StenmanU H, et al. Estimating the probability of cancer with several tumor markers in patients with colorectal disease [J].Oncology,2004,66(4):296-302.
42. OhiesMJ Erlich R. A review and update on cholangiocarcinoma [J]. Oncology, 2004 66(3):167-179
43. LiSM, YaoSK, Yan anuraN,etal Expression of Bel-2 and Bax in extrahepalic biliary tracl carcinoma andl clysplasia [J].World J Gastroenterol 2003,9(11):2579-2582
44. Marshall. J, Chen H,et al A phase I trial of a Bcl-2 antr sense(G3139)and weekly docelaxel in patients with advanced breast cancer anal other solid humors [J].Ann Oncol,2004,15(8):1274-1283
45. Dunbar SA, Vander zee CA, Oliver K G, et al. Quantitative, multiplexed detection of bacterial pathogens:DNA and protein applications of the Luminex LabMAP system [J]. J Microbiol Methods,2003,53(2):245-252.
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186. Koebel CM, VermiW, Swann JB, Zerafa N, Rodig SJ, Old LJ, et al. Adaptive immunity maintains occult cancer in an equilibrium state. Nature 2007;450:903-7.
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190. Shachaf CM, Kopelman AM, Arvanitis C,Karlsson A, Beer S, Mandl S, et al. MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature 2004;431:1112-7.
2. National Cancer Institute. Overview of the SEER Program. Available on the World Wide Web at http://seer.cancer.gov/about/. Accessed March 29,2006.
3. Nelson H, Petrelli N, Carlin A, et al. Guidelines 2000 for colon and rectal cancer surgery. J Natl Cancer Inst.2001;93:583-596.
4. Ward E, Jemal A, Cokkinides V, et al. Cancer disparities by race/ethnicity and socioeconomic status. CA Cancer J Clin 2004;54:78-93.
5. Ghafoor A, Jemal A, Cokkinides V, et al. Cancer statistics for African Americans. CA Cancer J Clin 2002;52:326-341.
6. Park JG. Cancer Incidence in Korea 1999-2001. Goyang:National Cancer Center; 2005.
7. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics:2007. CA Cancer J Clin.2007; 57:43-66.
8. Chen SL, Bilchik AJ. More extensive nod al dissection improves survival for stages I to III of colon cancer:a population-based study. Ann Surg.2006;244 602-10.
9. Kronberg U, Lopez-Kostner F, Soto G, Zuniga A, Wistuba I, Miranda V, et al. Detection of lymphatic micrometastases in patients with stages I and II colorectal cancer:impact on five-year survival. Dis Colon Rectum.2004;47:1151-7.
10. Rosenberg R, Friederichs J, Gertler R, Hoos A, Mueller J, Nahrig J, et al. Prognostic evaluation and review of immunohistochemically detected disseminated tumor cells in peritumoral lymph nodes of patients with pN0 colorectal cancer. Int J Colorectal Dis.2004; 19:430-7.
11. Tschmelitsch J, Klimstra DS, Cohen AM. Lymph node micrometastases do not predict relapse in stage II colon cancer. Ann Surg Oncol.2000;7:601-8.
12. Greene FL, Page DL, Fleming ID, Fritz A, Balch CM, Haller DG, et al. AJCC Cancer Staging Manual,6th ed. New York:Springer-Verlag; 2002. p.226-8.
13. Noura S, Yamamoto H, Ohnishi T, Masuda N, Matsumoto T, Takayama O, et al. Comparative detection of lymph node micrometastases of stage II colorectal cancer by reverse transcriptase polymerase chain reaction and immunohistochemistry. J Clin Oncol.2002;20:4232-41.
14. Bilchik AJ, Hoon DS, Saha S, Turner RR, Wiese D, DiNome M, et al. Prognostic impact of micrometastases in colon cancer:interim results of a prospective multicenter trial. Ann Surg.2007;246:568-75.
15. Benson AB 3rd, Schrag D, Somerfield MR, Cohen AM, Figueredo AT, Flynn PJ, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol.2004;22:3408-19.
16. Sun Jin Park, M.D.1, Kil Yeon Lee, M.D.1, PhD. and Si Young Kim, M.D., Ph.D.2 Clinical Significance of Lymph Node Micrometastasis in Stage I and II Colon Cancer, Cancer Res Treat.2008; 40(2):75-80.
17. Andre'T, Boni C, Mounedji-Boudiaf L, et al. Oxaliplatin fluorouracil and leucovorin as adjuvant treatment for colon cancer. N Engl J Med.2004; 350:2343-2351.
18. Baxter NN, Virnig DJ, Rothenberger DA, et al. Lymph node evaluation in colorectal cancer patients:a population-based Study. J Natl Cancer Inst.2005; 95:219-225.
19. Wong JH, Johnson DS, Hemmings D, et al. Assessing the quality of colorectal cancer staging:documenting the process in improving the staging of node-negative colorectal cancer. Arch Surg.2005;140:881-887.
20. Truong PT, Berthelet E, Lee J, et al. The prognostic significance of the percentage of positive/dissected axillary lymph nodes in breast cancer recurrence and survival in patients with one to three positive axillary nodes. Cancer. 2005;103:2006-2014.
21. Kim J, Cheong JH, Hyung WJ, et al. Predictors of long-term survival in pN3 gastric cancer patients. J Surg Oncol.2004;88:9-13.
22. Rodriguez Santiago JM, Munoz E, Marti M, et al. Metastatic lymph node ratio as a prognostic factor in gastric cancer. Eur J Surg Oncol.2005;31:59-66.
23. Berger AC, Sigurdson ER, LeVoyer T, et al. Colon cancer survival is associated with decreasing ratio of metastatic to examined lymph nodes. J Clin Oncol. 2005;23:8706-8712.
24. Jessup JM, Stewart A, Greene FL, et al. Adjuvant chemotherapy for stage III colon cancer:implications of race/ethnicity, age, and differentiation. JAMA.2005; 294:2703-2711.
25. Jemal A, Siegel R, Ward E, et al. Cancer statistics 2006. CA CancerJ Clin. 2006;56:106-130.
26. Swanson RS, Compton CC, Stewart AK, et al. The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined. Ann Surg Oncol. 2003;10:65-71.
27. Joseph NE, Sigurdson ER, Hanlon AL, et al. Accuracy of determining nodal negativity in colorectal cancer on the basis of the number of nodes retrieved on resection. Ann Surg Oncol.2003; 10:213-218.
28. Tiwari RC, Ghosh K, Jemal A, et al. A new method of predicting US and state-level cancer mortality counts for the current calendar year. CA Cancer J Clin 2004;54:30-40.
29. Oyama K, Terashima M, Takagane A, Maesawa C. Prognostic significance of peritoneal minimal residual disease in gastric cancer detected by reverse transcription-polymerase chain reaction. Br J Surg 2004;91:435-43..
30. Ozbas S, Dafydd H, Purushotham AD. Bone marrow micrometastasis in breast cancer. Br J Surg 2003;90:290-301.
31. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 2005;353:793-802.
32. Macadam R, Sarela A, Wilson J, MacLennan K, Guillou P. Bone marrow micrometastases predict early post-operative recurrence following surgical resection of oesophageal and gastric carcinoma. Eur J Surg Oncol 2003;29:450-4.
33. Seeliger H, Spatz H, Jauch KW. Minimal residual disease in gastric cancer. Recent Results Cancer Res 2003; 162:79-87.
34. Barak V, Goitre H, Panaretakis KW, et al. Clinical utility of cytokeratins as tumor markers[J]Clin Biochem.2004.37(7):529-540
35. Iwaya K.Mukai K. Accumulation of ubiquitin-conjugated cytokeratin fragments in tumor cells[J]Semin Cancer Biol.2005,15(4):309-318.
36. Alessandro R.Belluco C, Kohn E C. Proteomic approaches in colon cancer: promising tools for new cancermarkers and drugtarget discovery [J].Clin Colorectal Cancer.2005-4(6)396-402.
37. Chen Y D, Zheng S, Yu J K, et al. Artificial neural networks analysis of
surface-enhanced laser desorption/ionisation mass spectra of serum protein pattern distinguishes colorectal cancer from healthy population [J].Clin Cancer Res,2004,10(24):8380-8385.
38. Ward D G, Suggett N, Cheng Y, et al. Identification of serum biomarkers for colon cancer by proteomic analysis [J]Br J Cancer.2006.94(12):1898-1905.
39. Albrethsen J, Bogebo R, Cammeltofr S, et al. Upregulated expression of human neutrophil peptides 1.2 and 3 (HNP 1-3) in colon cancer serum and tumours:a biomarker study [J]. BMC Cancer.2005.5:8.
40. Turck N, Richest S, Cendry P, et al. Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells [J].Proteomics,2004,4(1):93-105.
41. Carpelan-Holmstrom M, Louhimo J, StenmanU H, et al. Estimating the probability of cancer with several tumor markers in patients with colorectal disease [J].Oncology,2004,66(4):296-302.
42. OhiesMJ Erlich R. A review and update on cholangiocarcinoma [J]. Oncology, 2004 66(3):167-179
43. LiSM, YaoSK, Yan anuraN,etal Expression of Bel-2 and Bax in extrahepalic biliary tracl carcinoma andl clysplasia [J]. World J Gastroenterol 2003,9(11):2579-2582
44. Marshall. J, Chen H,et al A phase I trial of a Bcl-2 antr sense(G3139)and weekly docelaxel in patients with advanced breast cancer anal other solid humors [J].Ann Oncol,2004,15(8):1274-1283
45. Dunbar SA, Vander zee CA, Oliver K G, et al. Quantitative, multiplexed detection of bacterial pathogens:DNA and protein applications of the Luminex LabMAP system [J]. J Microbiol Methods,2003,53(2):245-252.
46. Biagim RE,Sammons DL,Smith JP, etal. Simultaneous measurement of specific serum IgG responses to five select agents [J]. Anal Bioanal Chem, 2005,382(4):1027-1034.
47. Waterboer T, Sehr P, Pawlita M. Suppression non-specific binding in serological Luminex assays [J].J Immunol Methods,2006,309(1-2):200-204.
48. Pang S, Smith J, Onley D, et al. A comparability study of the emerging protein array platforms with established ELISA procedures [J]. J Immunol Methods,2005,302(1-2):1-12.
49. JAGER W. RIJKERS G T. Solid-phase and bead-based cytokine immunoassay: a comparison [J]. Methods,2006,38(4):294-303.
50. Ray CA. Bowsher RR. Smith WC, etal. Development validation and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum [J]. J Pharm Biomed Anal,2005,36(5): 1037-1044.
51. de JAGER W, PRAKKEN B J, BIJLSMA J W, et al. Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies [J]. J Immunol Methods, 2005,300(1-2):124-135.
52. Johanmsson A, Jonasson R, Dernfalk J, et al. Simultaneous detection of porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP technology [J]. Cytometry A,2006,69(5):391 - 395.
53. Moll R, Schiller D L, Franke W W. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns [J].J Cell Biol,1990, 111(2):567-580.
54. Dltnbar SA. Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection [J]. Clin Chim Acta,2006, 363 (1-2)73-82.
55. Lukacs Z, Dieich A, Ganschow R, et al. Simultaneous determination of HIV antibodies, hepatitis C antibodies, and hepatitis B antigens in dried blood spots-a feasibility study using a multi-analyte immunoassay [J]. Clin Chem Lab Med, 2005,43 (2):141 -145.
56. Flagella M, BUI S.Zheng Z, et al. A multiplex branched DNA assay for parallel quantitative gene expression profiling [J]. Anal Biochem,2006, 352(1):50-60.
57. Santoro R, Carlini M. Carboni F, et al. Delayed massive arterial hemorrhage after pancreaticoduodenectomy for cancer Managment of a life-threatening complication. Hepatogastroen-terology,2003.;50:2199-2204.
58. Mocellin S, Keilholz U, Rossi CR, et al. Circulating tumor cells:the leukemic phase of solid cancers [J].Trends Mol Med,2006,12(3):130 - 139
59. Molnar B, Sipos F, Galamb O, et al. Molecular detection of circulating cancer ceIIs.Role in diagnosis,prognosis and follow - up of colon cancer patient [J].Dig Dis,2003,21(4):320-325
60. Sato T, Harao M, Nakano S, et al. Circulating tumor cells deted by reverse transcription - polymerase chain reaction for carcinoembryonic antigen mRNA: distinguishing follicular thyroid carcinoma from adenoma[J]. Surgery,2005, 137(5):552 - 558
61. Friederichs J, Certler R, Rosenberg R, et al. Prognostic impact of CK-20-positive cells in peripheral venous blood of patients with gastrointestinal carcinoma[J].W orld J Surg,2005,29(4):422-428
62. Yamamoto O, Takahashi H, Hirasawa M, et al. Surfactant protein gene expressions for detection of lung carcinoma cells in peripheral blood [J]Respir Med,2005,99(9):1164 1174
63. Uen YH, Lin SR, Wu CH, et al. Clinical significance of MUCI and c-Met RT -PCR detection of circulating tumor cells in pabents with gastric carcinoma[J]. Clin Chim Acta,2006,367(1 - 2)55-61
64. Brooimans RA, de Leeuw N, Bontenbal M, et al. An immune-magnetic epithelial tumor cell enrichment model for minimalresidual disease detection of cytokeratin 8+ malignancies [J].J BiolRegul Homeost Agents,2005,19(1-2):84-91
65. Pachmann K, Clement K, Schneider CP, et al. Standardized quantification of circulating peripheral tumor cells from lung and breast cancer [J]. Clin Chem Lab Med,2005,43(6):617 - 627
66. Chen XM, Chen CY, Wang ZR, et al. Detection of micrometastasis of gastric carcinoma in peripheral blood circulation[J].W orld J Castroenterol,2004,10(6): 804-808
67. Bonmassar L, Massara MC, Cottarelli A, et al. Preclinical studieson detection of circulating melanoma cells in patients:telomerase as a recognition marker of malignancy[J].J Chemother,2004,16(5):479 - 486
68. Yao F, Guo JM, Xu CF, et al. Detecting AFP mRNA in peripheral blood of the patients with hepatocellular carcinoma, liver cirrhosis and hepatitis[J].Clin Chim Acta,2005,361(1 - 2):119 - 127
69. Guo J, Xiao B, Jin Z, et al. Detection of cytokeratin 20 mRNA in the peripheral blood of patients with colorectal cancer by immunomagnetic bead enrichment and real-time reverse transcaiptase-polymeras chain reaction[J].J Gastroenterol Hepatol,2005,20(8):1279-1284
70. Moreno JG, Miller MC, Gross S, et al. Circulating tumor cells predict survival in patients with metastatic prostate cancer[J] Urology,2005,65(4):713 718
71. Ma CJ,Hsieh JS, WangWH, et al. Multivariate analysis of prognostic determinants for colorectal cancer patients with high preoperative serun CEA levels:prognostic value of postoperative serum CEA levels [J]. Kaohsiung J Med Sci,2006,22(12):604-609
72. Grothev A,A comparison of XELOX with FOLFOX-4 as firsline trcatmcnt for metastatic colorectal cancer [J] Nat Clin Pract Oncol,2009,6 (1):10-11
1. Ghafoor A, Jemal A, Ward E, et al. Trends in breast cancer by race and ethnicity. CA Cancer J Clin 2003;53:342-355.
2. National Cancer Institute. Overview of the SEER Program. Available on the World Wide Web at http://seer.cancer.gov/about/. Accessed March 29,2006.
3. Nelson H, Petrelli N, Carlin A, et al. Guidelines 2000 for colon and rectal cancer surgery. J Natl Cancer Inst.2001;93:583-596.
4. Ward E, Jemal A, Cokkinides V, et al. Cancer disparities by race/ethnicity and socioeconomic status. CA Cancer J Clin 2004;54:78-93.
5. Ghafoor A, Jemal A, Cokkinides V, et al. Cancer statistics for African Americans. CA Cancer J Clin 2002;52:326-341.
6. Park JG. Cancer Incidence in Korea 1999-2001. Goyang:National Cancer Center; 2005.
7. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics:2007. CA Cancer J Clin.2007; 57:43-66.
8. Chen SL, Bilchik AJ. More extensive nod al dissection improves survival for stages I to III of colon cancer:a population-based study. Ann Surg.2006;244: 602-10.
9. Kronberg U, Lopez-Kostner F, Soto G, Zuniga A, Wistuba I, Miranda V, et al. Detection of lymphatic micrometastases in patients with stages I and II colorectal cancer:impact on five-year survival. Dis Colon Rectum.2004;47:1151-7.
10. Rosenberg R, Friederichs J, Gertler R, Hoos A, Mueller J, Nahrig J, et al. Prognostic evaluation and review of immunohistochemically detected disseminated tumor cells in peritumoral lymph nodes of patients with pN0 colorectal cancer. Int J Colorectal Dis.2004;19:430-7.
11. Tschmelitsch J, Klimstra DS, Cohen AM. Lymph node micrometastases do not predict relapse in stage II colon cancer. Ann Surg Oncol.2000;7:601-8.
12. Greene FL, Page DL, Fleming ID, Fritz A, Balch CM, Haller DG, et al. AJCC Cancer Staging Manual,6th ed. New York:Springer-Verlag; 2002. p.226-8.
13. Noura S, Yamamoto H, Ohnishi T, Masuda N, Matsumoto T, Takayama O, et al. Comparative detection of lymph node micrometastases of stage II colorectal cancer by reverse transcriptase polymerase chain reaction and
immunohistochemistry. J Clin Oncol.2002;20:4232-41.
14. Bilchik AJ, Hoon DS, Saha S, Turner RR, Wiese D, DiNome M, et al. Prognostic impact of micrometastases in colon cancer:interim results of a prospective multicenter trial. Ann Surg.2007;246:568-75.
15. Benson AB 3rd, Schrag D, Somerfield MR, Cohen AM, Figueredo AT, Flynn PJ, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol.2004;22:3408-19.
16. Sun Jin Park, M.D.1, Kil Yeon Lee, M.D.1, PhD. and Si Young Kim, M.D., Ph.D.2 Clinical Significance of Lymph Node Micrometastasis in Stage I and II Colon Cancer, Cancer Res Treat.2008; 40(2):75-80.
17. Andre'T, Boni C, Mounedji-Boudiaf L, et al. Oxaliplatin fluorouracil and leucovorin as adjuvant treatment for colon cancer. N Engl J Med.2004; 350:2343-2351.
18. Baxter NN, Virnig DJ, Rothenberger DA, et al. Lymph node evaluation in colorectal cancer patients:a population-based Study. J Natl Cancer Inst.2005; 95:219-225.
19. Wong JH, Johnson DS, Hemmings D, et al. Assessing the quality of colorectal cancer staging:documenting the process in improving the staging of node-negative colorectal cancer. Arch Surg.2005; 140:881-887.
20. Truong PT, Berthelet E, Lee J, et al. The prognostic significance of the percentage of positive/dissected axillary lymph nodes in breast cancer recurrence and survival in patients with one to three positive axillary nodes. Cancer. 2005;103:2006-2014.
21. Kim J, Cheong JH, Hyung WJ, et al. Predictors of long-term survival in pN3 gastric cancer patients. J Surg Oncol.2004;88:9-13.
22. Rodriguez Santiago JM, Munoz E, Marti M, et al. Metastatic lymph node ratio as a prognostic factor in gastric cancer. Eur J Surg Oncol.2005;31:59-66.
23. Berger AC, Sigurdson ER, LeVoyer T, et al. Colon cancer survival is associated with decreasing ratio of metastatic to examined lymph nodes. J Clin Oncol. 2005;23:8706-8712.
24. Jessup JM, Stewart A, Greene FL, et al. Adjuvant chemotherapy for stage III colon cancer:implications of race/ethnicity, age, and differentiation. JAMA.2005; 294:2703-2711.
25. Jemal A, Siegel R, Ward E, et al. Cancer statistics 2006. CA CancerJ Clin. 2006;56:106-130.
26. Swanson RS, Compton CC, Stewart AK, et al. The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined. Ann Surg Oncol. 2003; 10:65-71.
27. Joseph NE, Sigurdson ER, Hanlon AL, et al. Accuracy of determining nodal negativity in colorectal cancer on the basis of the number of nodes retrieved on resection. Ann Surg Oncol.2003; 10:213-218.
28. Tiwari RC, Ghosh K, Jemal A, et al. A new method of predicting US and state-level cancer mortality counts for the current calendar year. CA Cancer J Clin 2004;54:30-40.
29. Oyama K, Terashima M, Takagane A, Maesawa C. Prognostic significance of peritoneal minimal residual disease in gastric cancer detected by reverse transcription-polymerase chain reaction. Br J Surg 2004;91:435-43..
30. Ozbas S, Dafydd H, Purushotham AD. Bone marrow micrometastasis in breast cancer. Br J Surg 2003;90:290-301.
31. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 2005;353:793-802.
32. Macadam R, Sarela A, Wilson J, MacLennan K, Guillou P. Bone marrow micrometastases predict early post-operative recurrence following surgical resection of oesophageal and gastric carcinoma. Eur J Surg Oncol 2003;29:450-4.
33. Seeliger H, Spatz H, Jauch KW. Minimal residual disease in gastric cancer. Recent Results Cancer Res 2003; 162:79-87.
34. Barak V, Goitre H, Panaretakis KW, et al. Clinical utility of cytokeratins as tumor markers[J]Clin Biochem.2004.37(7):529-540
35. Iwaya K.Mukai K. Accumulation of ubiquitin-conjugated cytokeratin fragments in tumor cells[J]Semin Cancer Biol.2005,15(4):309-318.
36. Alessandro R.Belluco C, Kohn E C. Proteomic approaches in colon cancer: promising tools for new cancermarkers and drugtarget discovery [J].Clin Colorectal Cancer.2005-4(6)396-402.
37. Chen Y D, Zheng S, Yu J K, et al. Artificial neural networks analysis of surface-enhanced laser desorption/ionisation mass spectra of serum protein
pattern distinguishes colorectal cancer from healthy population [J].Clin Cancer Res,2004,10(24):8380-8385.
38. Ward D G, Suggett N, Cheng Y, et al. Identification of serum biomarkers for colon cancer by proteomic analysis [J]Br J Cancer.2006.94(12):1898-1905.
39. Albrethsen J, Bogebo R, Cammeltofr S, et al. Upregulated expression of human neutrophil peptides 1.2 and 3 (HNP 1-3) in colon cancer serum and tumours:a biomarker study [J]. BMC Cancer.2005.5:8.
40. Turck N, Richest S, Cendry P, et al. Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells [J].Proteomics,2004,4(1):93-105.
41. Carpelan-Holmstrom M, Louhimo J, StenmanU H, et al. Estimating the probability of cancer with several tumor markers in patients with colorectal disease [J].Oncology,2004,66(4):296-302.
42. OhiesMJ Erlich R. A review and update on cholangiocarcinoma [J]. Oncology, 2004 66(3):167-179
43. LiSM, YaoSK, Yan anuraN,etal Expression of Bel-2 and Bax in extrahepalic biliary tracl carcinoma andl clysplasia [J].World J Gastroenterol 2003,9(11):2579-2582
44. Marshall. J, Chen H,et al A phase I trial of a Bcl-2 antr sense(G3139)and weekly docelaxel in patients with advanced breast cancer anal other solid humors [J].Ann Oncol,2004,15(8):1274-1283
45. Dunbar SA, Vander zee CA, Oliver K G, et al. Quantitative, multiplexed detection of bacterial pathogens:DNA and protein applications of the Luminex LabMAP system [J]. J Microbiol Methods,2003,53(2):245-252.
46. Biagim RE,Sammons DL,Smith JP, etal. Simultaneous measurement of specific serum IgG responses to five select agents [J]. Anal Bioanal Chem, 2005,382(4):1027-1034.
47. Waterboer T, Sehr P, Pawlita M. Suppression non-specific binding in serological Luminex assays [J].J Immunol Methods,2006,309(1-2):200-204.
48. Pang S, Smith J, Onley D, et al. A comparability study of the emerging protein array platforms with established ELISA procedures [J]. J Immunol Methods,2005,302(1-2):1-12.
49. JAGER W. RIJKERS G T. Solid-phase and bead-based cytokine immunoassay: a comparison [J]. Methods,2006,38(4):294-303.
50. Ray CA. Bowsher RR. Smith WC, etal. Development validation and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum [J]. J Pharm Biomed Anal,2005,36(5): 1037 - 1044.
51. de JAGER W, PRAKKEN B J, BIJLSMA J W, et al. Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies [J]. J Immunol Methods, 2005,300(1 -2):124-135.
52. Johanmsson A, Jonasson R, Dernfalk J, et al. Simultaneous detection of porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP technology [J]. Cytometry A,2006,69(5):391 - 395.
53. Moll R, Schiller D L, Franke W W. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns [J].J Cell Biol,1990, 111(2):567-580.
54. Dltnbar SA. Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection [J]. Clin Chim Acta,2006, 363 (1-2)73-82.
55. Lukacs Z, Dieich A, Ganschow R, et al. Simultaneous determination of HIV antibodies, hepatitis C antibodies, and hepatitis B antigens in dried blood spots-a feasibility study using a multi-analyte immunoassay [J]. Clin Chem Lab Med, 2005,43 (2):141 -145.
56. Flagella M, BUI S.Zheng Z, et al. A multiplex branched DNA assay for parallel quantitative gene expression profiling [J]. Anal Biochem,2006, 352(1):50-60.
57. Santoro R, Carlini M. Carboni F, et al. Delayed massive arterial hemorrhage after pancreaticoduodenectomy for cancer Managment of a life-threatening complication. Hepatogastroen-terology,2003.;50:2199-2204.
58. Mocellin S, Keilholz U, Rossi CR, et al. Circulating tumor cells:the leukemic phase of solid cancers [J].Trends Mol Med,2006,12(3):130 - 139
59. Molnar B, Sipos F, Galamb O, et al. Molecular detection of circulating cancer ceIIs.Role in diagnosis,prognosis and follow - up of colon cancer patient [J].Dig Dis,2003,21(4):320-325
60. Sato T, Harao M, Nakano S, et al. Circulating tumor cells deted by reverse transcription - polymerase chain reaction for carcinoembryonic antigen mRNA: distinguishing follicular thyroid carcinoma from adenoma[J].Surgery,2005, 137(5):552-558
61. Friederichs J, Certler R, Rosenberg R, et al. Prognostic impact of CK-20-positive cells in peripheral venous blood of patients with gastrointestinal carcinoma[J].W orld J Surg,2005,29(4):422 - 428
62. Yamamoto O, Takahashi H, Hirasawa M, et al. Surfactant protein gene expressions for detection of lung carcinoma cells in peripheral blood [J]Respir Med,2005,99(9):1164 1174
63. Uen YH, Lin SR, Wu CH, et al. Clinical significance of MUCI and c-Met RT -PCR detection of circulating tumor cells in pabents with gastric carcinoma[J]. Clin Chim Acta,2006,367(1 - 2)55-61
64. Brooimans RA, de Leeuw N, Bontenbal M, et al. An immune-magnetic epithelial tumor cell enrichment model for minimalresidual disease detection of cytokeratin 8 malignancies [J].J BiolRegul Homeost Agents,2005,19(1-2):84-91
65. Pachmann K, Clement K, Schneider CP, et al. Standardized quantification of circulating peripheral tumor cells from lung and breast cancer [J]. Clin Chem Lab Med,2005,43(6):617-627
66. Chen XM, Chen CY, Wang ZR, et al. Detection of micrometastasis of gastric carcinoma in peripheral blood circulation[J].W orld J Castroenterol,2004,10(6): 804-808
67. Bonmassar L, Massara MC, Cottarelli A, et al. Preclinical studieson detection of circulating melanoma cells in patients:telomerase as a recognition marker of malignancy[J].J Chemother,2004,16(5):479 - 486
68. Yao F, Guo JM, Xu CF, et al. Detecting AFP mRNA in peripheral blood of the patients with hepatocellular carcinoma, liver cirrhosis and hepatitis[J].Clin Chim Acta,2005,361(1 - 2):119 - 127
69. Guo J, Xiao B, Jin Z, et al. Detection of cytokeratin 20 mRNA in the peripheral blood of patients with colorectal cancer by immunomagnetic bead enrichment and real-time reverse transcaiptase-polymeras chain reaction[J].J
Gastroenterol Hepatol,2005,20(8):1279-1284
70. Moreno JG, Miller MC, Gross S, et al. Circulating tumor cells predict survival in patients with metastatic prostate cancer[J] Urology,2005,65(4):713-718
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